Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of release factor expression using a translational negative feedback loop: A systems analysis RUSSELL BETNEY,1 ERIC DE SILVA,2 CHRISTINA MERTENS,1 YVONNE KNOX,1 J. KRISHNAN,2 and IAN STANSFIELD1,3 1University of Aberdeen, School of Medical Sciences, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom 2Chemical Engineering and Chemical Technology, Institute for Systems and Synthetic Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom ABSTRACT The essential eukaryote release factor eRF1, encoded by the yeast SUP45 gene, recognizes stop codons during ribosomal translation. SUP45 nonsense alleles are, however, viable due to the establishment of feedback-regulated readthrough of the premature termination codon; reductions in full-length eRF1 promote tRNA-mediated stop codon readthrough, which, in turn, drives partial production of full-length eRF1. A deterministic mathematical model of this eRF1 feedback loop was developed using a staged increase in model complexity. Model predictions matched the experimental observation that strains carrying the mutant SUQ5 tRNA (a weak UAA suppressor) in combination with any of the tested sup45UAA nonsense alleles exhibit threefold more stop codon readthrough than that of an SUQ5 yeast strain. The model also successfully predicted that eRF1 feedback control in an SUQ5 sup45UAA mutant would resist, but not completely prevent, imposed changes in eRF1 expression. In these experiments, the introduction of a plasmid-borne SUQ5 copy into a sup45UAA SUQ5 mutant directed additional readthrough and full-length eRF1 expression, despite feedback. Secondly, induction of additional sup45UAA mRNA expression in a sup45UAA SUQ5 strain also directed increased full-length eRF1 expression. The autogenous sup45 control mechanism therefore acts not to precisely control eRF1 expression, but rather as a damping mechanism that only partially resists changes in release factor expression level. The validated model predicts that the degree of feedback damping (i.e., control precision) is proportional to eRF1 affinity for the premature stop codon. The validated model represents an important tool to analyze this and other translational negative feedback loops. Keywords: release factor eRF1; SUP45; negative feedback; translation; ribosome; translation termination INTRODUCTION class I release factors, RF1 (recognizing UAA and UAG) and RF2 (recognizing UAA and UGA), have overlapping Cellular protein synthesis is a highly regulated process responsibility for recognizing the termination codons. A during which ribosomes sequentially translate an mRNA third, nonessential release factor, RF3, stimulates release template in a process that can be divided into the initiation, factor removal from the ribosome following termination elongation, and termination phases. Termination is triggered (Freistroffer et al. 1997; Gao et al. 2007). when the elongating ribosome encounters an in-frame stop The class I stop codon–recognizing release factors— codon, the signal for a translation release factor to bind and eRF1, RF1, and RF2—are essential for cell viability, a reflec- release the nascent polypeptide. In eukaryotes, the release tion of the fact that efficient recognition of the stop codon factor (RF) is a complex of two proteins: eRF1, a class I re- is crucial for accurate protein synthesis. If the release factor lease factor that can recognize each of the three stop codons; fails to recognize the stop codon efficiently, caused, for and eRF3, an essential GTPase that stimulates the efficiency example, by lowered RF cellular abundance, the extended of the termination process (Frolova et al. 1994; Stansfield pause will trigger at some level an alternative stop codon et al. 1995a; Zhouravleva et al. 1995). In prokaryotes, two readthrough process (Stansfield et al. 1995b; Bertram et al. 2001). Readthrough can be caused by mis-recognition of the stop codon by a natural suppressor tRNA. In yeast, 3Corresponding author Gln these ordinary tRNAs include the glutamine tRNAUUG E-mail [email protected] Gln Article published online ahead of print. Article and publication date are and tRNACUG , which, respectively, can decode UAA and at http://www.rnajournal.org/cgi/doi/10.1261/rna.035113.112. UAG via first-base G-U wobble (Pure et al. 1985; Edelman RNA (2012), 18:00–00. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2012 RNA Society. 1 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Betney et al. and Culbertson 1991). Such readthrough events will direct backgrounds (Moskalenko et al. 2003). The level of read- the synthesis of a potentially deleterious longer protein. through of the premature eRF1 nonsense codons is regulated Alternatively, longer pauses at bacterial stop codons, caused by a complex interaction between the suppressor tRNA by reduced RF activity at a weak stop codon, can trigger the efficiency (Stansfield et al. 1996; de Silva et al. 2010), the activity of tmRNA, a cellular emergency response to stalled eRF1 protein stability and activity, and the modulatory ribosomes that causes the incomplete protein to be targeted effect of the nonsense-mediated mRNA decay system that for degradation (Collier et al. 2002; Hayes et al. 2002a,b; acts to destabilize mRNAs carrying early stop codons Sunohara et al. 2004). (Chabelskaya et al. 2007; Kiktev et al. 2009). As in the While underabundance of release factors can allow read- case of the RF2 feedback loop, this system appears to be through events, their overexpression could potentially allow autoregulatory. erroneous recognition of ‘‘sense’’ codons as ‘‘stop,’’ causing Expression of functional eRF1 from the nonsense alleles, synthesis of a defective truncated protein. Bacterial release as with their RF2 counterpart, depends on a translation- factors can recognize near-cognate codons such as UGG with level negative feedback loop. However, the eRF1 control significant frequencies (Freistroffer et al. 2000). Further- circuit behavior relies on a straightforward two-way com- more, RF2 is able to trigger release of ribosomes and their petition between termination and stop codon readthrough, associated nascent peptides in cases in which a miscognate rather than the more complex three-way competition in the tRNA is located in the ribosomal P-site as a result of a prior bacterial RF2 circuit that additionally involves frameshift- translation error (Zaher and Green 2009). Arguably, RF2 ing. The simpler eRF1 case thus represents an ideal system overexpression could lead to inappropriate release of nascent with which to investigate the control properties of such polypeptides. translational negative feedback loops, the factors that Careful control over release factor expression is therefore influence their operation, and how robust they are to per- needed to ensure that these potent factors are maintained at turbation. The basic theoretical framework for a mathemat- appropriate levels, preventing the accidental expression of ical model of this system has been previously established shortened, or lengthened, polypeptides. It is thus intriguing (de Silva et al. 2010). In this study, we extend this by de- that RF2 expression is regulated at the translational level by veloping a fully parameterized mathematical model of the a negative feedback loop. The RF2 coding sequence is split eRF1 translational readthrough negative feedback loop and into two reading frames, separated by a UGA stop codon experimentally validate the predictions of the model. We (Craigen and Caskey 1986). The second, ORF2, is located show that the model successfully represents the establish- in the +1 frame relative to ORF1. Frameshifting is necessary ment of the feedback loop to maintain eRF1 production to translate RF2, and the efficiency of frameshifting is gov- and thus cell viability, and quantitatively predicts the level erned by a three-way competition between termination, of stop codon readthrough in these mutants. The model frameshifting, and readthrough (Curran and Yarus 1988; predicts that feedback loop constraint of eRF1 expression Adamski et al. 1993); it is proposed that a lowered RF2 level is regulated by the level of tRNA suppression, by the would trigger more frameshifting, and thus, RF2 synthesis premature stop codon nucleotide context, and by the (Craigen and Caskey 1986). However, although it is as- strength of the eRF1 promoter and thus level of mRNA. sumed that this system is autoregulatory, evidence support- The experimental data, analyzed in the context of the ing this is indirect and partly derived from studies in which validated model, suggest that for tight autoregulatory purified RF2 was added to in vitro translation reactions control, a premature stop codon in a good termination (Donly et al. 1990). The control properties of the RF2 au- context is required. togenous negative feedback circuit have thus not been fully explored. RESULTS An unusual relative of this regulatory system is found in yeast, where a series of nonsense alleles of the essential yeast Parameterization of a mathematical model eRF1 gene SUP45 have been isolated, all of which are viable describing eRF1 synthesis despite encoding a truncated eRF1 (Stansfield et al. 1996). Similar nonlethal nonsense alleles of the eRF3 gene (SUP35) A series of nonsense alleles of the yeast sup45